Di-c-GMP metabolism is important to bacterial biofilm formation and other virulence properties including intracellular survival of pathogens such as Salmonellae. Di-c-GMP is a second messanger that controls secretion and production of a variety of surface components of bacteria. Di-c-GMP is synthesized by specific enzymes containing a GGDEF amino acid motif and degraded by other proteins that contain an EAL protein motif. Specific binding proteins that contain domains termed PilZ domains then bind the second messenger and alter bacterial behavior and metabolism. We have developed preliminary data that indicates that FRET based assays can be used to precisely measure di-c-GMP binding. These proteins can also be used to measure synthesis and degradation of di-c-GMP by specific enzymes that synthesize or degrade the second message. This proposal will develop specfic high through put screens to define inhibitors of di-c-GMP synthesis, degradation, and binding that will be utilized to identify compounds that are candidate antibiotics based on this property. PUBLIC HEALTH RELEVANCE: Antibiotic resistance is an increasing problem and infections of medical devices, including catheters, is an important clinical problem. This grant will develop a screen for new drugs that will inhibit bacterial metabolism that will decrease the ability of bacteria to colonize medical devices and which could be used to block the virulence properties associated with extracellular protein secretion and biofilm formation.